1. Field of the Invention
Increasing genetic variability as a base for increased progress in animal breeding has been a long-term goal of agricultural research. Advances in molecular genetics suggest to us that it will soon be possible to introduce specific desirable genes into domestic animals.
The raw material for animal improvement is genetic variation. Recent advances in molecular genetics open the possibility for transferring new genetic information to the germ line of various species. Desirable genes could then be introduced into an improved line of domestic animals and these strains used for further improvement. For example, in chickens, egg production strains are highly susceptible to avian leukosis virus (ALV), and efforts to reduce the rate of congenital infection are underway by commercial breeders. Introduction of a dominant gene for resistance to ALV infection in available egg production strains would achieve this goal and thereby demonstrate that economically important new genes can indeed be introduced into the genome of a food animal species without disrupting the breeding program.
2. Description of the Prior Art
Insertion of foreign deoxyribonucleic acid (DNA) into the germ line of the mouse by microinjection of DNA into the male pronucleus of the newly fertilized ovum is now routine [Brinster et al., Proc. Natl. Acad Sci. USA 82: 4438-4442 (1985); Wagner, Can. J. Anim. Sci. 65: 539-552 (1985)]. Using similar techniques, genetic modification of economically important farm animals, such as pigs and sheep, has recently been reported [Hammer et al., Nature 315: 680-683 (1985)]. Because of the difficulty of manipulating new fertilized ova reviewed in Wagner, supra; Crittenden et al., Can. J. Anim. Sci. 65: 553-562 (1985); Freeman et al., World Poult. Sci. J. 41: 124-132 (1985); and Hughes et al., Poult. Sci. 65: 1459-1467 (1986)]similar results in the fowl have not been reported. However, natural insertion of genetic information into the chicken germ line has been occurring since the speciation of the chicken [Frisby et al., Cell 17: 623-634 (1979); Astrin et al., Cold Spring Harbor Symposium 44: 1105-1109 (1980); and Hughes et al., Cold Spring Harbor Symposium 44: 1077-1091 (1980)]. At least 22 endogenous viral genomes (proviruses) have been identified and characterized in the White Leghorn. Many more exist in other commercial lines of chickens [Hughes et al., Virology 108: 222-229 (1981); Gudkov et al., J. Gen. Virol. 57: 85-94 (1981)]. The presence of these proviruses suggests that there is a natural mechanism for proviral integration into the germ line. Presumably, retroviral infection of germ cells occurs on rare occasions. Spontaneous germ line insertion of murine leukemia virus has been detected in some strains of mice [Rowe et al., Proc. Natl. Acad. Sci. USA 77: 4871-4874 (1980); Herr et al., Nature 296: 865-868 (1982); Jenkins et al., Hybrid Mic. Cell 43: 811-819 (1985)]. That this can be done experimentally was shown first by Jaenisch, Proc. Natl. Acad. Sci. USA 73: 1260-1264 (1976) using retroviral infection of early mouse embryos and more recently by Van Der Putten et al., Proc. Natl. Acad. Sci. USA 82: 6148-6152 (1985) by infection of early mouse embryos with a retroviral vector.
Retroviruses offer substantial advantages for use as vectors both in cultured cells and in the intact animal [for a comprehensive review of the properties of retroviruses, see Weiss et al., RNA Tumor Viruses (1982)]. Retroviruses are the only viruses that behave as vectors in higher eukaryotes in nature. As such, it was reasonable to expect that these viruses could be readily adapted as vectors capable of accepting a wide variety of DNA sequences after suitable manipulations in the laboratory; a prediction that has been shown to be correct over the last few years. In addition, retroviruses can insert a DNA copy of their genomes into germ cells of a variety of species of higher eukaryotes in nature. Therefore, it is reasonable to expect to make use of these viruses, after inserting the desired sequences, as vectors for insertion of foreign DNA into the germ line. This has been done successfully both in the murine and in the avian systems. As vectors, these viruses offer other advantages as well.
Retroviral genomes are small, making it relatively easy to manipulate a cloned DNA copy of the genome. The viruses are efficient; in culture, essentially all of the cells can be infected. Most retroviruses are nonlytic; infection has little or no effect on cells in culture, and retroviruses exist that have no obvious deleterious effects on the intact animal. Since a DNA copy of the viral genome integrates into the host genome, the progeny of a single infected cell are all infected, and the provirus is in the same place in the genome of each of the progeny cells. Infection is self-limiting; each infected cell usually acquires 1-5 copies of the viral genome. Intervening sequences can be removed from genomic inserts cloned into a retroviral vector upon passage of the recombinant virus in cultured cells [Sorge et al., J. Mol. Appl. Genet. 1: 547-559 (1982); Shimotohno et al., Nature 299: 265-268 (1982)].
Unfortunately, there are also some disadvantages. Retroviruses, and the vectors derived from them, are relatively unstable. When a helper virus is present, there is extensive recombination, and even in the absence of a helper, internal rearrangements are frequently seen. The total size of the vector, including both the viral and nonviral sequences, is limited to about 10-11 kb [Shimotohno et al., Cell 26: 67-78 (1981); Sorge et al., J. Mol. Appl. Genet. 1: 547-559 (1982), supra; and Sorge et al., In Eukaryotic Viral Vectors, Y. Gluzman, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 127-133 (1982); Joyner et al., Mol. Cell. Biol. 3: 2180-2190 (1983); Norton et al., Mol. Cell. Biol. 5: 281-290 (1985)].
In our consideration for use of retroviral vectors intended for the insertion of genetic information into the germ line of chickens, it became apparent that the design of the vectors would be influenced by several important criteria. The ideal vector would be highly infectious, at least for the germ cells, so that a significant proportion of the progeny will have acquired the new genetic information. However, the vector should, upon entering the germ line, be incapable of further rounds of infection, so that it would become a stable part of the germ line, and so that in subsequent generations no reinfection of either germ line or somatic tissue would occur. The viral vector itself should have little or no adverse effect on the physiology of the host, and should not cause disease either in the host or in any other species. Finally, the vector should insert itself into many locations in the host genome. This may be important because the position may affect expression of the inserted information, and will be essential if a bird carrying two of more markers inserted by the same vector is to be created.